Arrington David D, Van Vleet Terry R, Schnellmann Rick G
Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Am J Physiol Cell Physiol. 2006 Dec;291(6):C1159-71. doi: 10.1152/ajpcell.00207.2006. Epub 2006 Jun 21.
Calpains, Ca(2+)-activated cysteine proteases, are cytosolic enzymes implicated in numerous cellular functions and pathologies. We identified a mitochondrial Ca(2+)-inducible protease that hydrolyzed a calpain substrate (SLLVY-AMC) and was inhibited by active site-directed calpain inhibitors as calpain 10, an atypical calpain lacking domain IV. Immunoblot analysis and activity assays revealed calpain 10 in the mitochondrial outer membrane, intermembrane space, inner membrane, and matrix fractions. Mitochondrial staining was observed when COOH-terminal green fluorescent protein-tagged calpain 10 was overexpressed in NIH-3T3 cells and the mitochondrial targeting sequence was localized to the NH(2)-terminal 15 amino acids. Overexpression of mitochondrial calpain 10 resulted in mitochondrial swelling and autophagy that was blocked by the mitochondrial permeability transition (MPT) inhibitor cyclosporine A. With the use of isolated mitochondria, Ca(2+)-induced MPT was partially decreased by calpain inhibitors. More importantly, Ca(2+)-induced inhibition of Complex I of the electron transport chain was blocked by calpain inhibitors and two Complex I proteins were identified as targets of mitochondrial calpain 10, NDUFV2, and ND6. In conclusion, calpain 10 is the first reported mitochondrially targeted calpain and is a mediator of mitochondrial dysfunction through the cleavage of Complex I subunits and activation of MPT.
钙蛋白酶是一种Ca(2+)激活的半胱氨酸蛋白酶，是一种参与多种细胞功能和病理过程的胞质酶。我们鉴定出一种线粒体Ca(2+)诱导的蛋白酶，它能水解一种钙蛋白酶底物（SLLVY-AMC），并被活性位点导向的钙蛋白酶抑制剂抑制，如钙蛋白酶10，这是一种缺乏结构域IV的非典型钙蛋白酶。免疫印迹分析和活性测定显示，钙蛋白酶10存在于线粒体外膜、膜间隙、内膜和基质部分。当在NIH-3T3细胞中过表达COOH末端绿色荧光蛋白标记的钙蛋白酶10时，观察到线粒体染色，并且线粒体靶向序列定位于NH(2)末端的15个氨基酸。线粒体钙蛋白酶10的过表达导致线粒体肿胀和自噬，而线粒体通透性转换（MPT）抑制剂环孢素A可阻断这种现象。使用分离的线粒体时，钙蛋白酶抑制剂可部分降低Ca(2+)诱导的MPT。更重要的是，钙蛋白酶抑制剂可阻断Ca(2+)诱导的电子传递链复合体I的抑制作用，并且鉴定出两种复合体I蛋白为线粒体钙蛋白酶10的靶标，即NDUFV2和ND6。总之，钙蛋白酶10是首个被报道的线粒体靶向钙蛋白酶，并且是通过切割复合体I亚基和激活MPT来介导线粒体功能障碍的介质。
